INVESTIGATOR?S DESCRIPTION: The broad, long-term objective of this proposal is to determine the role of P-selectin glycoprotein ligand- 1 (PSGL- 1) in regulating interactions between NK cells and leukocytes. Cell type and differentiation stage-specific post-translational modifications of a selectin binding domain located at the N-terminus of PSGL- 1 determine its relative specificity for P-, E- and L-selectins expressed on platelets, endothelial cells and leukocytes. We have discovered that NK cell maturation is accompanied by the modification of PSGL- 1 with a keratan sulfate-related lactosamine carbohydrate (the PEN5 epitope) that functions as an alternate L-selectin ligand. The aims of this proposal are: i) To determine the structural features of the PEN5 epitope that confer L-selectin binding, ii) To determine how interactions between PSGL-1 (+PEN5) and L-selectin affect NK cell function, iii) To determine whether ligation of PSGL- 1 (+PEN5) transmits signals to NK cells, and iv) To identify PSGL- 1 modifying enzymes that create the PEN5 epitope. We will determine the relative contribution of sialylation, fucosylation and sulfation to the creation of the alternate L-selectin ligand. We will also purify 0-glycans from PEN5+ cell lines and determine the structure of the PEN5 epitope. We will then determine the functional consequences of intracellular or intercellular interactions between PEN5 and L-selectin. Finally, we will use genetic screens to identify the enzymes required for assembly of the PEN5 epitope. The acquisition of an alternate L-selectin ligand by modification of PSGL- 1 during NK cell maturation has the potential to influence the way NK cells interact with leukocytes and/or target cells. Consequently, the overall goal of the proposed experiments is to determine how PEN5:L-selectin interactions influence NK cell function.